Renin is a proteolytic enzyme that, by releasing the angiotensins, plays a part in the regulation of the blood pressure. Human renin is a glycosylated protein having a molecular weight of approximately 40,000. Some structural characteristics of human renin are known. For example, the amino acid sequence of the polypeptide has been clarified by determining the complementary deoxyribonucleic acid (cDNA) that codes for renin. Renin is released in the kidneys from an inactive form, pro-renin, and then passes into the blood stream where it is to be found in concentrations of from 20 to 100 pg/ml. There, it brings about the cleavage of angiotensinogen (renin substrate) to form the decapeptide angiotensin I which in turn is cleaved by the so-called angiotensin-converting enzyme (ACE) in the lungs, kidneys and other organs to form the octapeptide angiotensin II. Angiotensin II raises the blood pressure both directly through arterial constriction and indirectly by releasing from the adrenal glands the hormone that causes sodium-retention, aldosterone, which is associated with an increase in the extracellular fluid volume. Angiotensin II is broken down by angiotensinases to the heptapeptide angiotensin III, which exhibits actions similar to those of angiotensin II, and to smaller, inactive fragments.
The treatment of certain forms of high blood pressure (hypertension) and cardiac insufficiency is possible by influencing the renin-angiotensin system. A lowering of blood pressure can be achieved by (a) inhibiting the action of angiotensin II (or III) on its receptors, (b) inhibiting the angiotensin-converting enzyme or (c) inhibiting the action of renin on angiotensinogen. Renin-inhibitors are, for example, pepstatin and pepstatin analogues, angiotensinogen analogues and renin antibodies. Such renin-inhibitors reduce the release of angiotensin I from angiotensinogen and thereby lessen also the concentration of the active peptide hormone angiotensin II. Compared with the known renin-inhibitors, the renin antibodies according to the invention have the advantage of inhibiting the action of renin quite specifically even in extremely low concentrations.
The use of antibodies in diagnosis and therapy was, until recently, severely limited in its range of application. Antibodies were obtained in very small amounts from animal serum in the form of a complex mixture of various proteins. Standardisation of the antibodies was not possible since each immunised individual animal, and even a single individual when immunised repeatedly, produces a serum with antibodies of a different composition in each case. By using a new technique developed by Kohler and Milstein [1] it has now become possible to obtain reproducibly in theoretically unlimited quantities antibodies in homogeneous form from cell cultures, that is to say so-called monoclonal antibodies. By fusion of suitable myeloma cells with antibody-producing lymphocytes from a donor immunised with antigen, hybridoma cells are produced which combine the ability to undergo unlimited cell division and unlimited growth in vitro with the production of a homogeneous antibody. Hence, it is possible to render independent the immune response of an organism to a specific antigen and to prepare monoclonal antibodies by continuous culturing of hybrid cells.
Although many examples of the preparation of specific monoclonal antibodies by the hybridoma technique have so far become known and the general procedure has been described in principle, with each new example specific problems arise that require adaptation of the technique to the particular case. Without such adaptation there is no certainty that the desired hybridoma cells will ever be formed, that they will be genetically stable and produce the desired monoclonal antibodies, and that the antibodies so prepared will have the desired specificity. The degree of success is influenced in principle by the type and purity of the antigen used for immunisation of the lymphocyte-donor, the method of immunisation, the technique of cell fusion, the procedure in the selection of suitable hybridoma cell lines and the way in which the monoclonal antibodies are isolated and purified.
Monoclonal antibodies that bind human renin are known. Simon et al. [2] describe a monoclonal antibody having a low affinity for human renin. Dzau et al. [3] describe monoclonal antibodies that bind human renin and simultaneously inhibit the enzymatic function thereof. For the determination of human renin in blood, basically, two different methods are used [4]. In one of these, the enzymatic activity of human renin in plasma is measured by determining the amount of angiotensin I released from angiotensinogen. A measure of the total amount of renin is then obtained by determining the enzymatic activity after activation of inactive plasma renin, for example by treatment with acid at pH 3 to 4. Although this indirect method allows the measurement of renin in low concentrations, it is subject to great uncertainties. In the other method, human renin is determined in immunoassays with the aid of the known monoclonal antibodies or with polyclonal antibodies from serum. This direct method, however, has not hitherto had sufficient sensitivity for a reliable measurement of the extraordinarily low concentrations of human renin in plasma. There is therefore a need for monoclonal antibodies that bind human renin considerably more strongly than do the known antibodies, so that they can be used in immunoassays to give an accurate determination of renin in human blood plasma. There is also a need for monoclonal antibodies that inhibit the action of the enzyme renin so efficiently that they can be used for the treatment of high blood pressure. The present invention represents a solution to this problem in that it provides monoclonal antibodies that bind human renin strongly and, at the same time, efficiently inhibit the enzymatic function thereof.